The plasma membrane ATPase P-glycoprotein (P-gp) confers multidrug resistance on tumor cells by the active transport of chemotherapeutic drugs out of the cells. P-gp is also present in certain normal human tissues, although its physiologic role is not well understood. One possible function of P-gp in normal cells is to protect the cells from naturally occurring carcinogens. Dietary factors such as the flavonoids have been shown to stimulate P-gp activity in tumor cells, however, the mechanism of stimulation is not known. Since the flavonoids are metal chelators, one possible mechanism would be to reduce free-radical production through a decrease in transition metals required for the Fenton reaction. In this project, we have examined the expression of P-gp in normal vascular endothelial cells and investigated the effect of dietary factors, including the flavonoid kaempferol and iron, on the activity of P-gp. Because of the anatomic location of the endothelium at the blood-tissue interface, it serves as a barrier to the uptake of substances by tissues. gp located in the endothelium could serve as part of this barrier and prevent tissue uptake of xenobiotics by mediating the transport back into the bloodstream of substances which enter the endothelial cells. Primary cultures of normal human endothelial cells were established from aorta, coronary arteries, and lung microvasculature. Using Western blot analysis for P-gp and Northern blot analysis for P-gp mRNA, we have established that P-gp is present in these cell types, with the highest expression in endothelial cells taken from the coronary arteries. The activity of P-gp is present in these cell types, with the highest expressio in endothelial cells taken from the coronary arteries. The activity of P- gp in coronary artery endothelial cells was monitored by measuring the ability of these cells to efflux the chemotherapeutic agents adriamycin and vinblastine. Both compounds were rapidly transported out of the cells. This efflux was inhibited by compounds known to inhibit P-gp, including verapamil. Thus, we have demonstrated that P-gp is present in endothelial cells and is functionally active. The dietary flavonoid kaempferol stimulated efflux, and increased intracellular iron inhibited efflux. These results are in agreement with previous experiments on P-gp activity in tumor cells. P-gp activity and its modulation by dietary factors in vascular endothelial cells may play an important role in the prevention of carcinogenesis.